Genetic Recombination and DNA Technology


     Genetic recombination is a process through which the genetic makeup of an organism is altered or changed in some way.  This alteration may be favorable, undesirable, or have no direct effect on an organism at all, depending upon the circumstances through which it occurs.  If recombination is favorable, it leads to genetic diversity by insuring that the organism has the ability to express new characteristics or traits which are not expressed by other members of the population, but can be passed to other members or offspring.  While recombination is similar to mutation in that it results in a change in the genotype of an organism, it is less likely to destroy the function of individual genes.
 

Homologous and Nonhomologous Recombination
      Recombination can occur either in an homologous or nonhomologous fashion.  In eukaryotic cells, homologous recombination occurs when the DNA which is exchanged is found in alternative forms of the same gene, called alleles, which lie on homologous eukaryotic chromosomes via the process of crossing over.  Crossing over occurs during the process of meiosis, when chromosomes line up.  Occasionally, chromatids from paired chromosomes overlap.  Their DNA can then be cleaved by endonuclease enzymes, then each piece swaps its place with the other and is enzymatically ligated (joined) to the new chromatid.  When the pairs are separated from one another, each chromosome is now genetically altered.  In prokaryotic cells, homologous recombination occurs when an endonuclease cleaves one DNA nucleoside leaving a free 3'-OH end, which then acts as a primer for DNA synthesis to occur.  The newly synthesized DNA nucleoside bonds to a corresponding region of the bacterial chromosome and forms a heteroduplex combination of single- and double-stranded DNA.  Such heteroduplex forms are catalyzed by enzymes which are coded for by recombination (rec) genes.

     Nonhomologous recombination does not involve specialized rec enzymes, but allows for the the joining of DNA molecules which have little similarity.  Unlike homologous recombination, this means that DNA fragments from one source, such as from a virus or bacterial plasmid, can be incorporated into a bacterial chromosome or the DNA of a eukaryotic cell.  If the nonhomologous DNA comes from a virus, a phenomenon called lysogenic conversion can occur, enabling the host cell to express viral genes.  For example, several bacteria species such as Staphylococcus aureus, Clostridium botulinum, and Corynebacterium diptheriae only become pathogenic after they have been invaded by a virus.

      Some cells contain small genetic elements of about 1000 nucleotides which have the ability to move or "jump" from one part of a chromosome to another.  These elements, called insertion sequences, do not appear to code for specific proteins but may serve as regulators for recombination of genetic material at specific sites, altering either promotor or structural genes.  In her study of corn genetics, Dr. Barbara McClintock discovered that other, larger transposable elements, called transposons, serve as structural genes and code for the production of specific proteins.  The insertion sites for these genes are random, thus their location can jump. Transposons are medically important in that they may code for antibiotic resistance factors in bacteria, and other microorganisms such as the pathogenic members of the protistan genera Trypanosoma and Plasmodium utilize such genes to create unique combinations of surface recognition glycoprotein compounds which allow them to overcome host defenses.
 

Gene Transfer

Transformation
     Frederick Griffith demonstrated in 1928 that living unencapsulated, nonpathogenic Streptococcus pneumoniae could become pathogenic if mixed with dead encapuslated pathogenic bacteria of the same species.  He called this process transformation, but was unaware of the chemical composition of the genetic material which was being passed between the dead and living cells.  In 1944, Avery, McCarty, and McCloud demonstrated that the material was DNA, not protein or RNA, both of which were hypothesized to be primary information storage molecules for cells.  Later research would show that in order for transformation to occur, the donor DNA must be released from a cell or cells after lysis, and that the live recipient bacteria must be competent, meaning that they must be capable of transporting the external DNA across their own plasma membrane or have specific receptor sites on the cell surface which enable them to do so.  Also, before any recombination of the donor and recipient DNA could occur, the recipient must be compatible with the host, thus the highest frequency of recombination occurs in cells which are closely related, such as different strains of the same species.  If tranformation does occur, the progeny of a transformed cell are said to be recombinants.  Examples of bacterial genera which can undergo transformation include Acinetobacter, Bacillus, Haemophilus, Neisseria, Rhizobium, Staphylococcus, and Streptococcus.
 
Transduction
     In 1952, Hershey and Chase demonstrated that viruses could transfer DNA to host cells through the radioactive labelling of bacteriophage protein and DNA.  The process of viral-mediated DNA exchange is called transduction.  Since DNA viruses such as some of the bacteriophages replicate within a host cell and host DNA is fragmented enzymatically during this process, some of the host cell DNA can be accidentally packaged by viral protein coats.  After these newly formed defective phages are released from the original host bacterium, they can infect new host cells and deliver their DNA, where recombination can take place.  This is not fatal to the host, and can confer upon it new characteristics.  If the genes carried by the defective phage are homologous to those of the recipient, the process is termed generallized transduction, and the recipient is said to be transduced.  If the genes are not homologous, such as those genes carried by temperate phages which are not defective, they may become incorporated into the bacterial chromosome.  If this DNA is copied from the host chromosome during the subsequent lytic cycle, it may also contain some of the host DNA, which can then be delivered to a new host.  This process is called speciallized transduction, and can result in the lysogenic conversion of species such as Corynebacterium diptheriae and Staphylococcus aureus.
 
 
Plasmids and the Process of Conjugation
     In 1950, Lederburg and Tatum discovered that some bacteria contain small, extrachromosomal genetic elements called plasmids which store genetic information not found in the chromosome.  Plasmids generally do not contain information which is necessary for the essential metabolic activities of the cell; however, they do contain genes which can determine the ability of the organism to pass genetic material to recipient cells.  Lederburg and Tatum found that some strains of E. coli contain the F (fertility) plasmid which enables these cells to form sex or conjugation pili.  Later, it was found that plasmids carry other characteristics as well.  E. coli, Shigella sp. and Salmonella sp. all have plasmids called R (resistance) factors which can give the cell the ability to produce enzymes which confer resistance to some antibiotics.  Some strains of E. coli also carry colicinogenic plasmids which code for the synthesis of colicin, a toxin which kills competing strains of E. coli.  Plasmids also can give bacteria the ability to produce toxins, and to degrade complex organic compounds such as hydrocarbons.

     If a bacterium has an F-plasmid, it is referred to as an F+ donor strain.  If the plasmid is not present, the bacterium is called an F- recipient.  During conjugation, The F+ cell forms a  conjugation pilus between itself and the recipient, which links the two cells and allows the commingling of their cytoplasm.  The F- plasmid is replicated within the donor, then passes through the pilus to the recipient, which now becomes an F+ cell.  If the donor plasmid recombines with the donor chromosome, a new type of cell called an Hfr (High frequency of recombination) cell is formed.  This cell can also participate in conjugation, however, a portion of the recombined chromosome is passed through the conjugation tube to the F- recipient.  After recombination, the F- cell is converted into a new Hfr cell.  The new cell, however, is generally will be incapable of conjugation itself, since only a portion of the genes contained in the original plasmid are passed via this process.

 Another phenomenon which can occur involves the formation of a new plasmid composed of genes which were located in the Hfr chromosome.  This plasmid contains both chromosomal genes and recombined plasmid genes.  After the new plasmid forms, the cell is called an F' cell rather than an Hfr cell.
 

Recombinant DNA Technology

Recombinant DNA
     Recombinant DNA technology involves the deliberate artificial union of DNA molecules from two or more different sources in a nonhomologous fashion.  This process allows researchers to give unrelated organisms the ability to express new phenotypes for a variety of different purposes.  For example, some individuals suffer from diabetes, which is the inability to control the level of sugar in the bloodstream.  This disorder arises from the lack of the hormone insulin, which is produced by special cells in the pancreas.  In past years, insulin used in the treatment of diabetes was derived from animals, since it was impossible to synthesize sufficient quantities of human insulin (humulin).  Unfortunately, many individuals suffered hypersensitive reactions to animal insulin.  With the advent of new molecular techniques for the insertion of foreign DNA into a bacterial chromosome or plasmid, it became possible to produce large quantities of humulin.  In brief, human insulin is composed of two polypeptides coded for by two different genes.  Researchers inserted these genes into the lac operon of E. coli, just after a nucleotide triplet which codes for the amino acid methionine.  This recombined bacterium began to produce humulin when in the presence of lactose, since the lac operon controls the production of enzymes necessary for the transport and catabolism of this sugar.

     The ability to insert foreign gene sequences into nonhomologous host DNA is derived from scientists' knowledge of a group of enzymes produced by some bacterial species as a protective measure against viral infection.  These enzymes, called restriction endonucleases cleave (cut) DNA along specific sequences of nucleotides.  If DNA is cleaved in such a way as to leave a few bases overhanging in palindromic sequences (sequences which read the same way in both directions; such as GGCATACGG and CCGTATGCC), the piece of DNA is said to have "sticky ends" since these overhanging portions of the 5' and 3' ends will form bonds (anneal) with any segment of DNA which has been cleaved to produce overhanging segments with complementary base pairs.  If, however, the restriction enzyme cleaves the molecule in such as way as to leave corresponding 5' and 3' ends bonded together in complementary fashion, the cut DNA molecule is said to have "blunt ends".  To bind a new or donor DNA fragment, it is necessary to establish artificial palindromy by binding multiple adenine (poly A) nucleotides to the donor and multiple thymine (poly T) to the recipient.

     Another technique which can be used to produce suitable DNA sequences for genetic engineering purposes is to utilize the properties of a viral enzyme called reverse transcriptase.  One of the problems inherent in the engineering of eukaryote DNA is that of split genes.  Recall that when transcription occurs in the eukaryotic nucleus, the usable portions of DNA, called exons, must first be separated from the nonsense portions called introns.  Rather than attempting to perform this process in-vitro (outside of the body), scientists purify the completed mRNA, then treat it with reverse transcriptase.  This enzyme binds DNA nucleotides to the mRNA template, producing a new DNA nucleoside.  DNA polymerase is used to complete the complementary nucleoside.  The newly formed gene can then be inserted into a bacterial plasmid.
 

The Polymerase Chain Reaction
    Sometimes it is necessary to have multiple copies of a sequence of DNA, if, for example, a sample of blood, semen, mtDNA (mitochondrial DNA), or some other DNA-containing substance is too small for proper analysis or genetic manipulation.  The technique called the polymerase chain reaction (PCR) can be used to amplify (clone) large numbers of the same DNA sequence.  To perform PCR, a piece of DNA is extracted from a  sample and purified.  This DNA is then heated to a temperature between 90o and 100o C, which causes the hydrogen bonds between complementary nucleosides to break without breaking the phosphodiester bonds between nucleotides.  Short segments of artificially produced DNA called oligonucleotides, which are complementary to the DNA nucleosides to be amplified, are added to the heated DNA sample, which is then allowed to cool.  These bind, or anneal, to the original DNA nucleosides.  Oligonucleotides act as primers for DNA replication, which is stimulated by the addition of individual nucleotides and DNA polymerase.  This process is repeated several times (called cycles), with each cycle resulting in a doubling of the number of identical copies of the original piece of DNA.  If PCR is performed properly, over one million copies of the orignal DNA strand can be produced within an hour.
Cloning
     Cloning is the asexual reproduction of genes containing recombined DNA.  Plasmids containing recombined gene sequences are often used as vectors for this technique, since they can be easily engineered and owing to the high reproductive rates of many bacterial species.  To clone a sequence of DNA, a vector is prepared containing the sequence along with another gene, which codes for the production of an enzyme for resistance to a particular antibiotic.  The vector is then inserted into bacterial cells via transformation or transduction, and the newly transformed or tran sduced cells are placed on media containing the antibiotic.  As the bacteria reproduce, many copies of the DNA sequence can be guaranteed, since only those cells resistant to the antibiotic will survive on the medium.  However, if foreign genes have been inserted by some other means such as unwanted viral infection or conjugation with other bacteria at the site of antibiotic resistance, the ability to block the antibiotic is lost.  Such insertional inactivation is a useful means to determine if foreign DNA is present in transformed cells.
Protoplast Fusion


     If the researcher wishes to recombine the entire DNA code (genome) from two or more cells, the technique of protoplast fusion can be performed.  To fuse prokaryote cells, the bacteria must first be stripped of their cell walls, then stored in a buffer solution which contains a high concentration of a solute such as sucrose.  The buffer prevents the protoplast cells from lysing due to osmotic pressure differences inside and outside of the cell membrane.  Protoplasts are then treated with polyethylene glycol which causes them to fuse.  The chromosomes of the fused cells then recombine.

     This technique can also be used with eukaryotic cells.  For example, cancer cells called mylenomas can be extracted from mouse tumor tissue.  These mutant tumor cells replicate rapidly (hyperplasia) and grow much larger than healthy cells of the same tissue type (hypertrophy).  Mylenoma cells can be fused in-vitro with antibody- producing B plasma cells, producing a new cell type called a hybridoma.  Hybridoma cells can be artificially cultured, grow quickly, and produce large quantities of specific immunoglobulins, called monoclonal antibodies, which when purified can be used to stimulate artificial active humoral immunity in patients suffering from various viral and bacterial- induced diseases.  This technique shows great promise in the treatment of many current human diseases.
 

Critical Thinking Question

     You and your research associates have just isolated a rare gene in a species of sea urchin (a multicellular eukaryotic marine animal) which codes for the production of an enzyme that blocks the entry of HIV into human cells.  Unfortunately, you have two problems; (A) you have isolated only a small sample of the genetic material from the one urchin you have been able to collect, and (B) gel electrophoresis has shown the sequence of DNA you need to be full of intron material.  Describe how you could overcome these problems, as well as how you could produce sufficient quantities of the enzyme to begin drug trials with HIV positive and full-blown AIDS patients.
 

  Test Yourself- Use this to test yourself about comcepts associated with recombination.
 
 
 

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